The broad goal of this proposal is to test whether microtubules are actively or passively involved in myofibril assembly in differentiating mouse myoblasts. Circumstantial evidence suggests that microtubules may participate in at least four temporally and/or spatially separate processes in differentiating striated muscle cells: cell elongation; myofibril assembly; sarcoplasmic reticulum (and T-tubule) elaboration; and myoblast fusion. If microtubules are required for myofibril assembly, independently of the other three processes then this supports the hypothesis that microtubules participate actively in myofibril assembly. Our proposal is designed to differentiate between the four microtubule- dependent events in myogenic differentiation by identifying microtubule- dependent molecules that participate in each of these processes. The broad, diverse and multifunctional family of kinesin-related microtubule motors is the most likely place to look for molecules that mediate distinct microtubule-dependent processes. During this funding period a subtractive cDNA expression library will be prepared from differentiating C2C12 myoblasts. Kinesin-related proteins that are specifically upregulated during myoblast differentiation will be identified by expression cloning from the subtractive library using anti-kinesin peptide antisera. The recovered putative myogenic kinesins will be analyzed for the ability to associate with myofibrillar proteins using column chromatography and pelleting assays. Null phenotypes engineered using antisense RNA and DNA will be used in combination with ectopic expression, motility assays, deletion constructs and observation of real-time dynamics by video microscopy to assign the myogenic kinesin(s) to one of the four aforementioned functional categories. Identification of a myogenic kinesin which associates with nascent myofibrils and is required for myofibril assembly will be direct evidence for active participation of microtubules in myofibrillogenesis. Furthermore, such a discovery would constitute the first direct evidence for cytoskeletal crosstalk during cytoplasmic reorganization. By the completion of the funding period, a new class of proteins involved in early stages of muscle development will be characterized and significant progress will be made toward understanding the role of microtubules in myofibrillogenesis. This proposal addresses a fundamental, yet neglected, area of muscle development which will help us better understand both clinical myopathies and also subcellular pattern formation in all biological systems.